Caveolin1 (Cav1) is an important component of the plasma-membrane microdomains, such as caveolae and lipid rafts, that are associated with AT1 and EGF receptors in certain cell types. An analysis of the interactions between Cav1 and other signaling molecules that mediate AT1R function was performed in Ang II- and EGF-stimulated hepatic C9 cells. This study demonstrated that cholesterol-rich domains mediate the actions of early upstream signaling molecules such as Src and intracellular calcium in cells stimulated by Ang II, but not by EGF, and that Cav1 has a scaffolding role in the process of MAPK activation. Furthermore, Cav1 phosphorylation by Ang II and EGF was regulated by calcium and Src. Phosphorylation of Cav1 and the EGFR by Ang II, but not ERK1/2 activation, are both dependent on calcium. The PI 3-kinase inhibitors, LY294002 and wortmannin, differentially modulated both Cav1 and EGF receptor activation by Ang II through calcium. These findings further demonstrate the importance of Cav1 in conjunction with receptor-mediated signaling pathways involved in cell proliferation and survival. It is clear that differential signaling pathways are operative in Ang II- and EGF- stimulated C9 cells, and that cholesterol-enriched microdomains are essential components in cellular signaling processes that are dependent on specific agonists and/or cell types.[unreadable] Relatively little is known about the protein-protein interactions that regulate the trafficking of the AT1R through the biosynthetic pathway. The membrane-proximal region of the cytoplasmic tail of the AT1R has been defined by site-directed mutagenesis studies as a site required for normal AT1R folding and surface expression. Based on yeast two-hybrid screening of a human embryonic kidney cDNA library with the AT1R carboxyl-terminal tail as bait, the Invariant chain (Ii) was identified as a novel receptor-interacting protein. This association was confirmed by co-immunoprecipitation and co-localization studies and the binding site for Ii on the AT1R carboxyl-terminal tail was localized to a region that has been identified as important for exit of the AT1R from the endoplasmic reticulum (ER), and is conserved in many G protein-coupled receptors. Transient co-expression of Ii with the AT1R in CHO cells consistently reduced the AT1R density at the cell surface. Furthermore, the interaction of Ii with the carboxyl-terminal tail of the AT1R promotes its retention in the ER and promotes its proteasomal degradation. These observations indicate that Ii and the AT1R become associated in the early biosynthetic pathway, and demonstrate that the Ii protein is a negative regulator of AT1R expression.[unreadable] In previous studies the molecular mechanism of the constitutive activity of AT1R mutants at position 111 was evaluated by molecular modeling. This involved a cascade of conformational changes in spatial positions of side chains along transmembrane helix 3 (TM3) from L112 to Y113 to F117, which in turn causes conformational changes in TM4 (residues I152 and M155) leading to its movement as a whole. This mechanism is consistent with the available data of site-directed mutagenesis, and with correct predictions of constitutive activity of mutants L112F and L112C. More recently, the network of inter-residue interactions within the transmembrane region of the AT1R was investigated by site-directed mutagenesis and molecular modeling studies. Mutagenesis was focused on residues Tyr292, Asn294, and Asn298 in transmembrane helix 7, and the conserved Asp74 in helix 2 and other polar residues. Functional interactions between pairs of residues were evaluated by determining the effects of single and double-reciprocal mutations on agonist-induced AT1R activation. Reciprocal mutations of Asp74/Asn294, as well as Ser115/Asn294, Ser252/Asn294, and Asn298/Ser115 caused additive impairment of function, suggesting that these pairs of residues make independent contributions to AT1R activation. In contrast, mutations of the Asp74/Tyr298 pair revealed that the D74N/N298D reciprocal mutation substantially increased the impaired inositol phosphate responses of the D74N and N298D receptors. Extensive molecular modeling yielded 3D models of the transmembrane region of the AT1R and the mutants as well as of their complexes with Ang II, which were used to identify possible mechanisms of impaired function of specific mutants. These data indicate that Asp74 and Asn298 are not optimally positioned for direct and strong interaction in the resting conformation of the AT1R. However, the balance of interactions between residues in helix 2 (such as D74) and helix 7 (such as N294, N295 and N298) of the AT1R is a crucial factor in determining their functional activity and levels of expression. [unreadable] Ang II promotes cell growth and proliferation, and has been implicated in several forms of tumorigenesis. The role of Ang II in prostate cancer was investigated in collaborative studies performed in the laboratory of Dr. Simon Louis in Melbourne, Australia. Ang II is present in the basal cell layer of the normal prostate gland and in benign prostatic hyperplasia (BPH), and stimulates prostate cell growth via the AT1R. Furthermore, AT1R blockers have been shown to reduce prostate-specific antigen and to inhibit prostate cancer cell growth. An analysis of Ang II expression in BPH and prostate cancer, including high grade prostatic intraepithelial neoplasia (HGPIN), showed its presence in basal epithelial cells in BPH and also in proliferating malignant cells in prostate cancer (Gleason grades 2-5), and in the cytoplasm of LNCaP, DU145, and PC3 prostate cancer cell lines. These data demonstrated Ang II staining in malignant cells in all grades of prostate cancer, and indicate that Ang II expression in non-basal epithelial cells is an early index of pre-malignant and malignant changes. In view of its mitogenic activity, it is probable that Ang II contributes to the growth and infiltration of malignant epithelial cells in the prostate. Furthermore, based on the observation by Baker et al. (2006) that elevated levels of cytoplasmic Ang II can increase cell proliferation via a non-AT1R mechanism, it is possible that angiotensin converting enzyme (ACE) inhibitors would also be of value in the treatment of prostate cancer by reducing intracellular Ang II formation.[unreadable] In collaboration with Dr. Sue Goo Rhee, formerly of the NIH, the mechanism responsible for the Ang II-induced production of reactive oxygen species in non-phagocytic cells was investigated in HEK293 and CHO cells reconstituted with the AT1R, NADPH oxidase 1 (Nox1), Nox organizer 1 (Noxo1), and Nox activator 1 (Noxa1). Stimulation of the reconstituted cells with Ang II caused a substantial increase in superoxide production relative to the constitutive level mediated by the complex of Nox1, Noxo1, and Noxa1. This demonstrated that Nox1 is activated by cell-surface receptor-mediated signaling, and that the AT1R is coupled to Nox1. Expression of AT1R mutants showed that interaction of the receptor with G proteins, but not that with beta-arrestin or proteins (Jak2, phospholipase C-gamma1, SHP2) that bind to the carboxy-terminal region of the AT1R, was necessary for Ang II-induced superoxide production. Evaluation of the effects of constitutively active alpha subunits of G proteins and of various pharmacological agents suggested that signaling by a pathway comprising the AT1R, Galphaq/11, phospholipase C-beta, and protein kinase C was largely, but not exclusively, responsible for Ang II-induced activation of the Nox1-Noxo1-Noxa1 complex in the reconstituted cells. Contributions of Galpha12/13, phospholipase D,and PI3-kinase to Ang II-induced superoxide generation were also suggested, whereas the small GTPase, Rac1, and the EGF receptor do not appear to participate in this action of Ang II.